1. Field of the Invention
The present invention relates to a technique for performing crystallization of macromolecules, and more particularly, it relates to a technique for performing crystallization of various biological macromolecules such as proteins by employing a semiconductor substrate or the like whose valence electrons are controlled.
2. Description of the Background Art
For understanding specific properties and functions in various types of biological macromolecules such as protein and complexes thereof, detailed steric structures thereof are indispensable information. From the basic chemical viewpoint, for example, information on the three-dimensional structure of protein or the like serves as the basis for understanding the mechanism of function appearance in a biochemical system by an enzyme or hormone. Particularly in the fields of pharmaceutical science, genetic engineering and chemical engineering among industrial circles, the three-dimensional structure provides information indispensable for rational molecular design for facilitating drug design, protein engineering, biochemical synthesis and the like.
As a method of obtaining three-dimensional steric structural information of biological macromolecules at atomic levels, X-ray crystal structural analysis is the most cogent and high-accuracy means at present. The analytic speed is remarkably improved by rapid improvement of arithmetic processing speed of computers in addition to reduction of measuring times and improvement of measuring accuracy due to recent hardware improvement of X-ray light sources and analyzers. The three-dimensional structures are conceivably going to be clarified by the main stream of this method also from now on.
In order to decide the three-dimensional structure of a biological macromolecule by X-ray crystal structural analysis, on the other hand, it is indispensable to crystallize the target substance after extraction and purification. At present, however, there is neither technique nor apparatus which can necessarily crystallize any substance when applied, and hence crystallization is progressed while repeating trial and error drawing on intuition and experience under the present circumstances. A search under an enormous number of experimental conditions is necessary for obtaining a crystal of a biological macromolecule, and crystal growth forms the main bottleneck in the field of the X-ray crystallographic analysis.
Crystallization of a biological macromolecule such as protein is basically adapted to perform a treatment of eliminating a solvent from an aqueous or anhydrous solution containing the macromolecule thereby attaining a supersaturated state and growing a crystal, similarly to the case of a general low molecular weight compound such as inorganic salt. As typical methods therefor, there are (1) a batch method, (2) dialysis and (3) a gas-liquid correlation diffusion method, which are chosen depending on the type, the quantity, the properties etc. of a sample.
The batch method is a method of directly adding a precipitant eliminating hydration water to a solution containing a biological macromolecule for reducing the solubility of the biological macromolecule and converting the same to a solid phase. In this method, solid ammonium sulfate, for example, is frequently used. This method has such disadvantages that the same requires a large amount of solution sample, fine adjustment of a salt concentration and pH is difficult, skill is required for the operation, and reproducibility is low. As shown in FIG. 46, for example, the dialysis, which overcomes the disadvantages of the batch method, is a method of placing a solution 572 containing a biological macromolecule inside a sealed dialytic tube 571 for continuously changing the pH etc. of a dialytic tube outer liquid 573 (e.g., a buffer solution) and making crystallization. According to this method, the salt concentrations of the inner and outer liquids and the pH difference are adjustable at arbitrary speeds, and hence the conditions for crystallization are easy to find out. As shown in FIG. 47, for example, a gas-liquid correlation diffusion method among the diffusion methods is a technique of placing a droplet 582 of a sample solution on a sample holder 581 such as a cover glass and placing this droplet and a precipitant solution 584 in a closed container 583, thereby slowly setting up equilibrium by evaporation of volatile components therebetween. Furthermore, as shown in FIG. 48, a liquid phase-liquid phase diffusion method among the diffusion methods is a technique of placing a droplet 592 of a mother liquor containing a target substance and a droplet 591 of a precipitant on a substrate 590 at a space of about 5 mm, and forming a thin liquid flow 593 with the point of a needle or the like therebetween. Mutual diffusion is occurred through the liquid flow 593, and crystallization is facilitated. This diffusion method has such an advantage that the amount of the solution may be extremely small as compared with the batch method or the like.
However, there are various problems in crystallization of a biological macromolecule such as protein as described above, in the present circumstances.
First, it has been difficult to obtain a crystal of excellent crystallinity or a large-sized single crystal. It is considered that this is because a biological macromolecule is readily influenced by gravity since its molecular, weight is generally large and causes convection in a solution (e.g., F. Rosenberger, J. Cryst. Growth, 76, 618 (1986)). Namely, the biological macromolecule or a formed fine crystal nucleus precipitates by its own weight, whereby convection of the solution around the molecules or the nucleus is caused. Also around the formed crystal surface, the concentration of the molecules is decreased and local convection of the solution takes place. Due to the convection in the solution generated in the aforementioned manner, the formed crystal moves in the solution, and moreover the layer for supplying the molecules by diffusion in the periphery of the crystal is remarkably reduced. Thus, the crystal growth rate can be reduced, or anisotropic growth can take place on the crystal plane, so that crystallization can be hindered.
A large amount of solvent (mainly water) (.gtoreq.50 volume %) is contained in a biological macromolecule crystal, dissimilarly to crystals of other substances. This solvent is disorderly and readily movable in the intermolecular clearances of the crystal. Although the molecules are gigantic, further, there is little wide-ranging intermolecular packing contact in the crystal and only slight molecule-to-molecule contact or contact by hydrogen bond through water molecules is present. Such a state is also the factor hindering crystallization.
Further, a biological macromolecule is extremely sensitive to the conditions employed for crystallization. While the biological macromolecule is stabilized in the solvent by interaction between individual molecular surfaces, charge distributions on the molecular surfaces, particularly conformation of amino acids in the vicinity of the molecular surfaces etc., extremely vary with the environment, i.e., pH, ionic strength and temperature of the solution, and type and dielectric constant of the buffer solution, and the like. Therefore, the crystallization process has been a multi-parameter process in which complicated various conditions are entangled with each other, and it has been impossible to establish a unific technique which is applicable to any substance. As to protein, crystallization of hydrophobic membrane protein is extremely difficult at present although it is biochemically extremely important as compared with water-soluble protein, and very few examples of the hydrophobic membrane protein have succeeded in crystallization and analysis of high resolution.
Further, the obtained biological macromolecule is generally in a very small amount. For example, protein such as enzyme is generally extracted from cells or the like and purified, while the amount of the sample finally obtained for crystallization is generally extremely small since its content is small. It is said that the concentration of a biological macromolecule in a solution should be about 50 mg/ml for performing crystallization. Therefore, repeated crystallization experiments (screening) under various conditions should be carried out as to a solution of an amount as small as possible.
While the amount of the sample may be small in the diffusion methods as described above, optimum conditions for crystallization must be found out by varying the salt concentration, the pH etc. of the precipitant over a wide range to obtain a crystal of high quality. In this case, the conditions can be found out only by trial and error. Further, a glass substrate for forming a droplet of the sample thereon readily causes mass generation of unnecessary crystal nuclei. In order to suppress this, surface treatments such as surface polishing and a water-repellent treatment should be previously performed
As described above, crystallization of biological macromolecules such as protein and complexes thereof forms the most significant bottleneck for the X-ray crystal structural analysis since the same has heretofore been progressed while repeating trial and error, although this is an important process in science and industry. Therefore, it is necessary to hereafter understand the basic principle of crystallization and develop a crystallization technique that is applicable to any molecule.